Vibrio cholerae bacteria cause the severe diarrheal disease cholera, which afflicts an estimated 5 million people annually worldwide. NIH has recently designated global health as a priority research area. When ingested, V. cholerae detect signals in the human gut that induce the virulence factor production required for causing disease. Expression of V. cholerae virulence factors absolutely requires ToxT protein, the subject of this proposal. The ToxT crystal structure was recently solved, which facilitates a new level of analysis that will be exploited. Recent work identified bicarbonate as an important signal that is sensed by V. cholerae and enhances ToxT activity to induce virulence. This is the first known example of a host signal that induces production of V. cholerae virulence factors. However, there are significant gaps in our knowledge regarding: i) how bicarbonate is sensed, ii) how it enters the bacteria, and iii) how it regulates ToxT activity. There are further significant gaps in our understanding of the fundamental mechanisms ToxT uses to activate V. cholerae virulence such as: i) whether ToxT functions as a monomer or dimer, ii) how individual virulence genes activated by ToxT are expressed at different levels and times during infection, and iii) how ToxT interacts with the transcriptional machinery. To address all of these gaps, three major hypotheses will be tested: 1) Direct binding by ToxT of the host signal bicarbonate increases its binding affinity for DNA sites at virulence gene promoters. 2) Bicarbonate affects ToxT dimerization, which is important for activation of some virulence promoters. 3) The timing and level of expression of individual virulence genes is determined by differences in bicarbonate-regulated ToxT binding. To test these hypotheses, a combination of biochemical and genetic techniques will be used. Direct binding of bicarbonate by ToxT will be assessed. The genes required for bicarbonate to induce V. cholerae virulence will be identified using a sophisticated FACS-based genetic screen. The two most likely mechanisms for the effect of bicarbonate on ToxT, ToxT dimerization and/or increased DNA binding affinity, will be tested both genetically and using purified proteins and DNA. The higher order structure of active ToxT, both in solution in the presence and absence of DNA and within the V. cholerae cytosol, will be determined. The question of how promoter architecture impacts differences in the expression levels and timing of individual virulence genes will be resolved by measuring DNA binding affinity biochemically and correlating with timing and magnitude of gene expression. This new information will give us a comprehensive understanding of how a host signal induces V. cholerae virulence. The proposed research will significantly advance the field by closing many gaps in our knowledge of V. cholerae pathogenesis and could identify new therapeutic targets for future research. Furthermore, this work on the most important V. cholerae virulence regulator, ToxT, will contribute to studies of proteins similar to ToxT in many other pathogens. Thus the proposed work could establish a new paradigm for control of bacterial pathogenesis.